Project Summary/Abstract Our goal is to develop novel quantitative super-resolution microscopy (SRM) methodologies to investigate the changes in higher-order chromatin structure that occur during embryonic development in single cells and at nanometer resolution. As a model system, we will use the developmentally-regulated silencing of an entire chromosome. Female mammals inactivate one of their two X chromosomes by X-chromosome Inactivation (XCI) to ensure dosage compensation between sexes. In undifferentiated embryonic stem cells (ESCs) both X- chromosomes are active (Xa) and XCI is induced upon differentiation. The hallmark of XCI is the expression and the cis spreading of the long non-coding RNA (lncRNA) Xist from its locus along the entire future inactive X- chromosome (Xi). The three-dimensional (3D) organization of the Xi is dependent on Xist RNA as the Xist knockout re-establishes the Xa configuration. Profound structural differences have been described between the Xa and Xi by imaging and genomics methods. The Xa is organized like autosomes with distinct segregation of active and inactive regions, while Xi is described as a monotonous structure, where compartmentalization is lost. Although the state of the Xi and Xa are well defined, the steps of higher-order structural changes that occur as the Xa turns into the Xi remain fully unexplored. Similarly, very little is known about the changes in chromosome conformation and its relationship to gene silencing and the spatial distribution of Xist on the chromosome during XCI and whether it's stochastic or deterministic for chromatin reorganization as the X becomes heterochromatic. Genomics approaches showed that at the onset of XCI, Xist spreads from its site of transcription to 28 linearly distal sites on the X (entry sites) due to their spatial proximity, while it covers the entire chromosome when the Xi is fully formed. By employing 3D-Structured Illumination Microscopy (3D-SIM), we showed that Xist displays ~ 100 distinct foci throughout the Xi-territory. These findings raise the question of how Xist exerts its chromosome-wide function on the Xi when it is constrained to only 100 clusters and lead to the hypothesis that whole chromosome coverage predicted by genomics approaches is a result of stochastic or highly dynamic associations or cross-linking effects due to the highly compacted Xi-chromatin. Additionally, the dynamics of Xist clusters and their real-time association to chromatin have not been addressed. Understanding theses mechanism will inform on how genomes alter their structure during development or in disease and how chromatin structure begets gene function. Here, we will unveil how the X chromosome structure changes as it inactivates and how Xist associates with the changing X, by developing unique SRM imaging approaches, offering an unprecedented opportunity to explore the dynamics of 3D chromatin changes during development and the role of lncRNAs in this process. To achieve these goals, we will: 1) delineate higher-order chromatin changes that occur on the X-chromosome during initiation of XCI and identify the chromatin targets of Xist RNA at the single cell level, 2) monitor Xist RNA dynamics in living cells at nanometer resolution.